The objective of this work is to clarify the genetic and biochemical mechanisms producing impaired oxidative decarboxylation of branched chain alpha-ketoacids. Branched chain alpha-ketoacid dehydrogenase has been solubilized and partially purified from rat liver. Characterization studies revealed that a single enzyme complex was acting to decarboxylate the three branched chain alpha-ketoacids. Four cofactors are necessary for activity; thiamine pyrophosphate, Mg 2ion CoASH and NAD ion and thiamine pyrophosphate also appears to play a stabilizing role in complex association. These studies will continue with cultured skin fibroblasts comparing normal cells with those exhibiting decreased enzyme function due to Maple Syrup Urine disease, an autosomal recessively inherited inborn error of emtabolism. Studies with cultured cells will center around fluorescent immunohistochemical techniques of protein identification in these cells using antibodies to the purified enzyme complex. Information gained will clarify the genetics and biochemistry of human branched chain alpha-ketoacid decarboxylation and provide a rationale for thiamine therapy in Maple Syrup Urine disease as a means of preventing mental and physical retardation resulting from this genetic abnormality. BIBLIOGRAHIC REFERENCES: Elsas, L. J., Wheeler, F.B., Danner, D.J. and DeHaan, R.L. (1975) J. Biol. Chem. 250:9380-9390. Amino Acid Transport in Aggregates of Cultured Chicken Heart Cells: Effect of Insulin. Elsas, L. J. and Danner, D.J. Effect of Thiamine on Normal and Mutant Human Branched Chain alpha-Ketoacid Dehydrogenase. U. S.-Japan Seminar on Thiamine. C. Gubler and M. Fujirawa Eds., Wiley Interscience, New York (1976).